Study on the expression of human lysozyme in oviduct bioreactor mediated by recombinant avian adeno-associated virus1

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Abstract

Due to its antimicrobial properties and low toxicity, human lysozyme (hLYZ) has broad application in the medical field and as a preservative used by the food industry. However, limited availability hinders its widespread use. Hence, we constructed a recombinant avian adeno-associated virus (rAAAV) that would specifically express hLYZ in the chicken oviduct and harvested hLYZ from the egg whites of laying hens. The oviduct-specific human lysozyme expression cassette flanked by avian adeno-associated virus (AAAV) inverted terminal repeats (ITRs) was subcloned into the modified baculovirus transfer vector pFBX, and then the recombinant baculovirus rBac-ITRLYZ was generated. The recombinant avian adeno-associated virus was produced by co-infecting Sf9 cells with rBac-ITRLYZ and the other 2 baculoviruses containing AAAV functional genes and structural genes, respectively. Electron microscopy and real-time PCR revealed that the recombinant viral particles were generated successfully with a typical AAAV morphology and a high titer. After one intravenous injection of each laying hen with 2 × 1011 viral particles, oviduct-specific expression of recombinant human lysozyme (rhLYZ) was detected by reverse transcription-PCR. The expression level of rhLYZ in the first wk increased to 258 ± 11.5 μg/mL, reached a maximum of 683 ± 16.4 μg/mL at the fifth wk, and then progressively declined during the succeeding 7 wk of the study. Western blotting indicated that the oviduct-expressed rhLYZ had the same molecular weight as the natural enzyme. These results indicate that an efficient and convenient oviduct bioreactor mediated by rAAAV has been established, and it is useful for production of other recombinant proteins.

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